winkler et al bbe supplementary material - media. · PDF fileWinkler et al., Mechanism of BBE,...

Winkler et al., Mechanism of BBE, supplementary material

1

A concerted mechanism for berberine bridge enzyme

Andreas Winkler, Andrzej Łyskowski, Sabrina Riedl, Martin Puhl,

Toni M. Kutchan, Peter Macheroux & Karl Gruber

Supplementary Material

Contents: Supplementary Figures (7 figures) 2

Supplementary Table 9

Supplementary Discussion 10

Supplementary Methods 13


2

Figure S1: Schematic representation of the structure of BBE from Eschscholzia californica

(A), of glucooligosaccharide oxidase from Acremonium strictum (B, PDB entry: 1zr6), 6-

hydroxy-D-nicotine oxidase from Arthrobacter nicotinovorans (C, PDB-entry: 2bvf) and of

aclacinomycin oxidoreductase from Streptomyces galilaeus (D, PDB-entry: 2ipi). The

similarities between the four structures are evident from r.m.s-deviations of 1.5 to 1.6 Å for

330 to 370 superimposed Cα-atoms. In A, B and D the flavin cofactor is bi-covalently

attached to the protein by a histidine (to C8α) and a cysteine (to C6). In the 6-hydroxy-D-

nicotine oxidase from Arthrobacter nicotinovorans (C) the cysteine attachment is missing.


3

Figure S2: (a) Two perpendicular views of the Fo-Fc density map of the isoalloxazine ring

system plus His104 and Cys166 in BBE_tet contoured at 5.0 σ. (b) Active site of BBE in the

tetragonal crystal form showing His459 in two alternate conformations. (c) Two

perpendicular views of the omit density map of the isoalloxazine ring system plus His104 and

Cys166 in BBE_mon contoured at 3.0 σ. The observed density was interpreted as a

superposition of intact FAD (orange) and an intermediate product of hydrolysis in the

isoalloxazine ring system (white)1. Two partially occupied water molecules are shown as blue

spheres. (d) Time dependent degradation of the cofactor obtained during incubation in the

dark at RT in 50 mM Tris/HCl pH 9.0. Spectra were recorded after 0, 23, 65, 120 and 191 h

and are represented by the solid, dashed, dotted, dash-dotted and dash-dot-dotted line,

respectively. Spectra were corrected for protein precipitation during the long incubation time.

(e) Schematic representation of the observed degradation process under alkaline conditions.


4

Figure S3: (S)-Reticuline binding. (a) Stereo view of the Fo-Fc electron density map of the

bound (S)-reticuline in the active site contoured at 3.0 σ . The amino acids involved in the

covalent attachment of the cofactor were omitted for clarity. (b) Stereo view of the active site

cleft of BBE. The flavin cofactor (blue) and the substrate (S)-reticuline (yellow) are shown in

a ball-and-stick representation together with a cartoon representation and the semi-transparent

molecular surface of the protein.


5

Figure S4: Comparison of wild-type enzyme and the E417Q variant. Spectral changes

observed during single turnover experiments carried out with wild-type BBE (a) and the

E417Q mutant protein (b). 11 and 6 µM protein solutions, respectively, were incubated with

an excess of (S)-reticuline under anoxic conditions and spectra were recorded at various time

points. The time-course starts with the solid line and ends at the dash-dot-dotted line spanning

0.1 and 160 seconds for the wild-type and mutant protein, respectively. (c) Comparison of the

native UV-Vis absorbance spectra of wild-type (solid) and E417Q (dotted) normalized at 445

nm. (d) Kinetic traces of cofactor reduction monitored at 445 nm upon mixing ~200 µM (S)-

reticuline with ~ 9 µM protein solution (wild-type - solid, E417Q - dotted) in an anoxic

environment.


6

Figure S5: Spectral changes accompanying (R,S)-laudanosine oxidation to 2-methyl-3,4-

dihydropapaverinium (13). ~ 3 µM wild-type BBE was incubated with 500 µM of the

substrate analog shown in the inset. The first spectrum is represented by a solid line and

spectral changes as indicated by the arrows were recorded over a period of 2 hours (dash-dot-

dotted line).


7

Figure S6: Side-product identification. HPLC analysis revealed that conversion of (S)-

reticuline (7.4 min) by E417Q results in a mixture of two products (13.8 and 14.3 min) in a

ratio of ~1:2, respectively. The major product peak represents the standard product of the

BBE catalyzed reaction [(S)-scoulerine, 14.3 min]. The additional compound produced during

this conversion was found to elute at the same time as an authentic reference for (S)-

coreximine (13.8 min). The numbers in brackets represent rounded elution times of the

substances, which are also indicated in more detail on top of each peak in the elution profiles.

The insets show the chemical structures of the two products formed.


8

Figure S7: Alignment of the sequences of berberine bridge enzyme from Eschscholzia

californica (Swissprot: P30986), Δ1-tetrahydrocannabinolic acid synthase (TrEMBL:

Q8GTB6) and cannabidiolic acid (CBDA) synthase (TrEMBL: A6P6V9) from Cannabis

sativa. The multiple sequence alignment was prepared using TCoffee (http://www.igs.cnrs-

mrs.fr/Tcoffee/) taking into account secondary structure elements derived from the BBE

structure. The active site residues Glu417, His459 and Tyr106 as well as the residues His104

and Cys166, which form the bi-covalent linkage of the flavin cofactor in BBE, are marked.


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Table S1: Data collection and refinement statistics.

BBE_mon BBE_tet BBE_compl

Data collection

Space group C2 P41212 C2

Cell dimensions

a, b, c (Å) 99.91, 94.84, 63.98 68.52, 68.52, 246.30 98.94, 93.65, 63.16

α, β, γ (°) 90.0, 100.3, 90.0 90.0, 90.0, 90.0 90.0, 100.6, 90.0

Resolution (Å) 25.0-1.65

(1.68-1.65)* 30.0-2.05

(2.09-2.05)* 26.0-2.80

(2.85-2.80)*

Rsym 0.071 (0.278) 0.096 (0.678) 0.115 (0.424)

I / σI 26.5 (4.6) 22.9 (2.8) 7.8 (2.2)

Completeness (%) 98.7 (95.9) 96.2 (96.9) 88.9 (91.3)

Redundancy 3.7 (3.5) 6.7 (7.0) 2.9 (2.9)

Refinement

Resolution (Å) 25.0-1.65 30.0-2.05 26.0-2.8

No. reflections 69824 36664 12513

Rwork / Rfree 0.1548/0.1864 0.1845/0.2256 0.1877/0.2406

No. atoms

Protein 4098 3997 3938

Cofactor/substrate/sugars 193 106 163

Water 780 463 18

B-factors

Protein 18.7 33.1 43.3

Cofactor/substrate/sugars 24.0 44.3 57.5

Water 36.0 42.0 30.9

R.m.s. deviations

Bond lengths (Å) 0.009 0.002 0.003

Bond angles (°) 1.3 0.7 0.8

*Values in parentheses are for highest-resolution shell.


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Supplementary Discussion

X-ray structure analysis

In all three structures (Table S1), 14 to 18 residues at the C terminus are missing in the

electron density. The first visible residues at the N terminus are Asp26 (in BBE_mon), Ala23

(in BBE_tet) and Ala21 (in BBE_compl). The numbering scheme is according to the

published sequence2, which includes a 23 residue signal peptide at the N terminus. Residues

in the structures with numbers smaller than 24 originate from remnants of the yeast α-factor

which was introduced for an optimal expression in Pichia pastoris3. Clear electron density

was observed for sugar moieties attached to residues Asn38 and Asn471, which were both

predicted as glycosylation sites. While only the first N-acetyl-glucosamine was visible at the

latter position, a hexasaccharide (Nag2-Man4) was placed into the electron density extending

from Asn38 in BBE_mon.

The molecular structure comprises two domains: an FAD binding domain (consisting of two

N-terminal α/β-subdomains and a C-terminal, mostly α-helical stretch) and a central α/β-

domain with a seven stranded, anti-parallel β-sheet forming the substrate binding site.

According to an analysis using the MSDssm-server4, the closest structural neighbors of BBE

are members of the p-cresol methylhydroxylase (PCMH) superfamily (Fig. S1):

glucooligosaccharide oxidase from Acremonium strictum5 (r.m.s.d.: 1.66 Å for 369 aligned

Cα-atoms), 6-hydroxy-D-nicotine oxidase from Arthrobacter nicotinovorans6 (1.67 Å/334)

and aclacinomycin oxidoreductase from Streptomyces galilaeus7 (1.51 Å/363).

While the electron density of FAD was as expected for a nearly planar isoalloxazine ring

system in the tetragonal crystal form (Fig. S2), the density of the isoalloxazine ring in the

monoclinic structure exhibited uncommon features that were interpreted as originating from a

superposition of an intact flavin molecule and a 4a-spirohydantoin1,8 degradation product

(Fig. S2). The relatively long incubation times at pH 8.5 necessary for the growth of the

monoclinic crystals led to a partial bleaching of the intensely yellow crystallization drops and

of the crystals themselves. Control experiments under similar alkaline conditions also showed

a time dependent degradation of the flavin spectrum (Fig. S2). In the substrate complex, the

electron density around the flavin also indicates the presence of the degradation product, but

to a much smaller extent compared to the high resolution monoclinic structure. This finding is

consistent with the time elapsed between crystallization setup and data collection (five days

vs. ten weeks).


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The tetragonal crystals appeared very quickly and the diffraction dataset was collected about

two days after setting up crystallization, whereas the monoclinic crystals took weeks to grow

to usable sizes and the high resolution dataset was collected ten weeks after the setup. The

degradation of the cofactor under alkaline conditions is also interesting with respect to the

intracellular localization of BBE. Under physiological conditions BBE – with its relatively

high pH optimum9 – supposedly catalyzes its reaction in endoplasmic reticulum derived

alkaline vesicles until they fuse with the central vacuole10,11. Since no activity of BBE is

expected under the acidic conditions of the vacuole there seems to be no evolutionary

pressure to ensure cofactor stability over prolonged periods at the conditions required to

effectively catalyze its reaction. This contrasts to other members of the group of bi-covalently

flavinylated oxidases, which have an extracellular localization12,13 and therefore require a

better long term stability.

The conformations of active site residues are not significantly altered upon complexation with

(S)-reticuline. The side chain of Trp165, however, for which only weak electron density was

observed in the unbound structures, becomes better ordered in the complex and interacts with

the phenolic ring of the isoquinoline moiety.

Kinetic analysis

A detailed comparison of the biochemical properties of the E417Q to wild-type enzyme is

presented in Fig. S4 demonstrating that the UV/Vis-absorbance of the FAD cofactor is not

affected by the replacement of glutamate, which can be expected due to its relatively distant

positioning (> 6 Å). Reduction of the cofactor by (S)-reticuline is accompanied by similar

spectral changes for both the wild-type enzyme and the E417Q mutant protein, showing a

fully reduced spectrum with a broad absorption maximum around 400 nm and also the fully

oxidized spectrum of FAD is virtually identical between the two proteins.

In the case of H459A, the effects observed on kred and kcat are only up to two-fold indicating

that this amino acid does not play a major role in catalysis. On the other hand, the influence of

the Y106F replacement is more pronounced leading to a 10-fold decrease of the turnover rate

in steady-state measurements and a similar decrease of the reductive rate observed in single

turnover rapid reaction experiments of the reductive half reaction. Interestingly, the turnover

rates for the latter two muteins are affected even though their reductive rates (kred) are much

higher (H459A) or in the same range (Y106F) as the steady-state turnover (kcat) of wild-type

BBE. Since turnover is limited by the oxidative-half reaction of the cofactor in the case of the

wild-type protein14, this indicates that reoxidation might also be affected by the introduced


12

amino acid substitutions. Even though a detailed mechanism of the reoxidation of flavoprotein

oxidases still awaits to be elucidated, the proximity of Tyr106 and His459 to the C4a position

of the isoalloxazine ring system might point towards a role during the oxidative half

reaction15. Analysis of the cofactor reoxidation revealed that these two amino acid

substitutions indeed have an effect on the oxidative rate (Table 1, main text). In the case of

H459A, the second order rate constant for reoxidation is only half of that of the wild-type

protein, which is in line with the reduction of the turnover rate by a factor of two. The same

reduction in oxygen reactivity is observed for Y106F. In this case, however, the decrease in

the rate of cofactor reoxidation cannot explain the observed reduction for the steady-state

turnover (Table 1, main text) indicating that other processes are relevant for catalytic

efficiency.

Due to the close positioning of both Tyr106 and His459 to the newly formed carbon-carbon

bond, both amino acids could also play a role in providing the appropriate environment for

product rearomatization prior to its dissociation or the regeneration of the active site for a new

enzymatic cycle.

Enzymatic conversion of substrate analogs

The NMR-data of the reaction product obtained in a conversion of the substrate analog 8 are

as follows: 1H NMR (500 MHz, D2O): δ 8.69 (s, 1H), 7.35 (s, 1H), 7.12 (s, 1H), 4.00 (s, 3H),

4.00 (t, J = 8.2 Hz, 2H), 3.92 (s, 3H), 3.73 (s, 3H), 3.24 (t, J = 8.2 Hz, 2H); 13C NMR (125

MHz, D2O): δ 164.9, 156.6, 147.8, 132.9, 117.2, 115.0, 111.3, 56.5, 56.2, 49.5, 46.8, 24.7.

These data clearly show the generation of a double bond in conjugation to the aromatic ring

by the appearance of an additional aromatic proton signal at 8.69 ppm and by the downfield

shift of the N-methyl proton signals due to the positive charge on the nitrogen atom (4.0 ppm

compared to 2.82 ppm in case of 8).


13

Supplementary Methods

Chemicals and reagents

(S)-Reticuline and (S)-coreximine were from the natural product collection at the Donald

Danforth Plant Science Center. (R,S)-Laudanosine was from Extrasynthese. Oligonucleotides

were from VBC-Biotech and purified by polyacrylamide gel electrophoresis. Site-directed

mutagenesis was carried out using the QuikChange® XL kit (Stratagene). Standard chemicals

were obtained from Sigma-Aldrich.

Crystallization

Wild-type BBE was expressed in Pichia pastoris and purified as described before3. The

enzyme was crystallized at room temperature using the sitting drop vapor diffusion method

with drops of 1 μL protein solution (~30 mg mL-1 in 50 mM Tris/HCl, 150 mM NaCl, pH 9.0)

plus 1 μL reservoir solution. Diffraction quality crystals were obtained with 0.2 M MgCl2 and

30 % (w/v) PEG-4000 in 0.1 M Tris/HCl pH 8.5. Depending on the protein sample (from

different Pichia fermentations) monoclinic or tetragonal crystals appeared (Table S1). While

the latter grew overnight after microseeding, monoclinic crystals took weeks to grow to

usable sizes. For cryoprotection the crystals were transferred to a solution containing 25%

glycerol before flash-cooling in liquid nitrogen. For the soaking with substrate this solution

additionally contained 20 mM (S)-reticuline and the crystal was incubated for roughly one

minute.

Structure determination

A first complete dataset to 2.05 Å resolution was collected from a monoclinic crystal at our

in-house rotating anode generator (Cu-Kα-radiation, λ=1.5418 Å) and was used for initial

structure solution. Subsequently, datasets BBE_mon and BBE_compl were collected at

beamlines X13 (λ=0.8148 Å) and X11 (λ=0.8010 Å) at the EMBL/DESY Hamburg, whereas

dataset BBE_tet was again collected at our in-house source (Table S1). In all cases, data

reduction involved the programs DENZO and SCALEPACK16 as well as software from the

CCP4 suite17.

The structure was solved by molecular replacement with the program PHASER18 using the

first monoclinic dataset. The search model – obtained from the CaspR server19 – was a

truncated homology model of BBE mainly comprising the FAD binding domain and was


14

based on the structures of glucooligosaccharide oxidase from Acremonium strictum (PDB

entry: 1zr6)5, 6-hydroxy-D-nicotine oxidase from Arthrobacter nicotinovorans (PDB-entry:

2bvf)6 and aclacinomycin oxidoreductase from Streptomyces galilaeus (PDB-entry: 2ipi)7.

The overall sequence identity of these templates with BBE is about 24%. Based on this initial

phase information, the major part of the structure was automatically built using PHENIX20

and ARP/wARP21. Missing amino acids as well as the FAD cofactor could be placed in the

resulting clear difference electron density. The other structures were either solved by

molecular replacement or by rigid body refinement using the partially refined model.

The structures were further refined using the program PHENIX20. Model building and fitting

steps involved the graphics program Coot22 using σA-weighted 2Fo-Fc and Fo-Fc electron

density maps23. Rfree-values24 were computed from 5% randomly chosen reflections not used

throughout the refinement. In the higher resolution structures, water molecules were placed

automatically into difference electron density maps and were retained or rejected based on

geometric criteria as well as on their refined B-factors. In the complex structure only few well

defined water molecules were manually placed into the electron density. Details of the data

collection, processing and structure refinement are summarized in Table S1. Coordinates and

structure factors have been deposited with the Protein Data Bank under the accession numbers

3D2H (monoclinic), 3D2J (tetragonal) and 3D2D (substrate complex).

Site-directed mutagenesis

Mutagenesis was carried out with the expression vector pPICZα BBE-ER described

previously3 in order to alter amino acids potentially involved in the catalytic mechanism of

BBE. Based on the structure three amino acids in the proximity of the catalytic center were

chosen for an initial analysis. Tyr106, Glu417 and His459 were changed to Phe, Gln and Ala,

respectively, using polymerase chain reaction-based mutagenesis. The primer pairs used for

mutagenesis as described in QuikChange® XL Site-directed mutagenesis kit (Stratagene)

consisted of a sense primer (sequences indicated below) and the complementary antisense

primer. 5'-GAAGTGGTGGTCATAGTTTTGAAGGATTATCTTACACTTCTG-3' for

Y106F, 5'-CGAAGTGGTACAAGATTAATGGTTCAATATATAGTTGCCTGGAATC-3'

for E417Q and 5'-

CCAAGACTTGGGTATGTTAATGCTATTGATCTTGATCTTGGAGGGATA-3' for

H459A with the underlined codon representing the changed position. Introduction of the

expected mutation was verified by plasmid sequencing. Mutein expression and purification

followed the same procedure as for the wild-type.


15

The ratio A280/A445 was virtually unaffected for both the H459A and E417Q mutant proteins

when compared with wild-type BBE indicating that the proteins are fully loaded with

cofactor. In the case of Y106F, this ratio was slightly changed due to spectral changes

indicative of 4a-spirohydantoin formation during the purification process8. It can thus be

concluded that this amino acid affects stability of the cofactor. However, since protein

concentrations were estimated based on the known extinction coefficient for the FAD cofactor

of wild-type BBE3, only the concentration of intact Y106F entered the calculations of

turnover rates.

Turnover-rate determination

For steady-state kinetic analysis the conversion of (S)-reticuline to (S)-scoulerine was

monitored by high performance liquid chromatography. Separation of the two substances was

carried out on an Atlantis® dC18 column (5 μm, 4.6×250 mm, Waters) using an isocratic

elution with 60 % MeOH/40 % 10 mM ammonium bicarbonate buffer, pH 7.0, for 11

minutes. Reaction mixtures consisted of a total volume of 200 μL with 100 µM (S)-reticuline

in 100 mM Tris/HCl pH 9.0 – the pH optimum of BBE9 – and an enzyme concentration

adjusted for each mutein in order to obtain a linear conversion rate during the initial phase of

the reaction.

Side-product identification

Identification of the additional product peak observed during turnover assays of the E417Q

mutein was performed by comparing the elution time with an authentic standard of the

suspected side-product (S)-coreximine. Slightly different conditions were used for a better

separation of the two products compared to standard activity assays. Isocratic elution with 50

% MeOH/50 % 10 mM ammonium bicarbonate buffer, pH 7.0, was carried out on an

Atlantis® dC18 column (5 µm, 4.6×150 mm, Waters) over 20 minutes.

Transient-kinetics

Determination of the reductive rates was performed as reported in a previous study14.

Apparent rate constants for the reductive half-reaction were measured at five different

concentrations of (S)-reticuline - 25, 50, 75, 125, 200 and 300 μM, and the concentration of

each mutein was ~10 μM (all concentrations are corrected for dilution after mixing in the flow

cell). The reductive rate was calculated from a hyperbolic fit to the apparent rates at all

substrate concentrations and represents the rate under saturating substrate concentrations.


16

Spectral changes during single-turnover experiments were monitored with a KinetaScanT

diode array detector (MG-6560) attached to the stopped-flow device (SF-61DX2 from TgK

Scientific). Rates for the oxidative half-reaction were measured by mixing substrate reduced

enzyme solution with air-saturated buffer (21 % oxygen) as described previously14.

Enzymatic conversion of substrate analogs

The substrate analog 6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline was converted

by BBE and the product identified by NMR. To that end, 2.47 mg of the latter substance were

dissolved in D2O containing 10 mM Tris/Cl and adjusted to a pD ~ 9. Conversion of the

substrate was initiated by addition of 0.5 mg recombinant BBE and monitored by TLC. After

completion of the reaction the protein was heat precipitated and after centrifugation the

supernatant was used for 1H and 13C NMR spectroscopy. The overlap of an assumed triplet

with a singlet at 4.00 ppm showing a total 5-H integral was confirmed by COSY

spectroscopy, which showed the coupling of this signal with the second triplet at 3.24 ppm.

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